The long-term goal of the work is to expand understanding of secretin family receptors and to facilitate the development of drugs acting at these receptors. This proposal focuses on structural characterization of the secretin receptor with selected studies also directed toward the VIP receptor. The general hypothesis guiding this work is that detailed insights into the structure of the prototypic secretin receptor and into the molecular mechanisms of its ligand-binding and activation will provide important leads to strategies for the rational development of receptor-active drugs. Aim 1 is designed to explore the hypothesis that the amino-terminal domain of this receptor, with its conserved cysteine residues and disulfide bonds, folds into a unique stable structure. This will be investigated using biochemical, molecular biological, and cell biological approaches leading to the definition of specific bonds that can be used as constraints for molecular modeling. Aim 2 is designed to test the hypothesis that the amino-terminal domain of the secretin receptor provides a critical binding pocket for the natural peptide ligand. Studies will use the complementary approaches of affinity labeling with a series of unique photolabile probes and receptor mutagenesis to define molecular approximations between residues within the ligand and the receptor. Aim 3 is designed to test the hypothesis that secretin family receptors are activated by a tethered transduction mechanism in which structurally and functionally independent receptor domains interact in a unique way. Experimental focus for this series of studies will be shifted to the body of the receptor. Mutagenesis will be performed on residues that are conserved within the family and likely play an architectural support role, as well as on residues that can be correlated with the selectivity of binding and activation of the receptors. The minimal receptor structure consistent with constitutive activity will be determined using a series of truncation mutants. Additionally, a novel mode of reverse affinity labeling with a photolabile probe incorporated specifically into the receptor will be performed. This will directly explore molecular proximity between distinct domains within the receptor.